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Phylogenetic studies of genomic data can provide valuable opportunities for evaluating evolutionary timescales and drivers of rate variation. These analyses require statistical tools based on molecular clocks. We present ClockstaRX, a flexible platform for exploring and testing evolutionary rate signals in phylogenomic data. It implements methods that use information from gene trees in Euclidean space, allowing data transformation, visualization, and hypothesis testing. ClockstaRX implements formal tests of the dimensionality reducibility of the Euclidean space of rates, and for identifying loci and branches that have a large influence on rate variation. Using simulations to evaluate the performance of the methods implemented, we find that inferences about rates can be strongly influenced by the overall amount of rate variation in the data, the shared patterns of among-lineage rate heterogeneity across groups of loci, and missing data. In an analysis of phylogenomic data from birds, we find a higher rate of evolution in introns compared with exons across all lineages. In addition, passerine taxa are highlighted as having unique patterns of genomic evolutionary rates compared with other avian lineages. Drawing on these results, we recommend careful exploratory analyses and filtering before performing phylogenomic analyses using molecular clocks.

The germline mutation rate determines the pace of genome evolution and is an evolving parameter itself1. However, little is known about what determines its evolution, as most studies of mutation rates have focused on single species with different methodologies2. Here we quantify germline mutation rates across vertebrates by sequencing and comparing the high-coverage genomes of 151 parent–offspring trios from 68 species of mammals, fishes, birds and reptiles. We show that the per-generation mutation rate varies among species by a factor of 40, with mutation rates being higher for males than for females in mammals and birds, but not in reptiles and fishes. The generation time, age at maturity and species-level fecundity are the key life-history traits affecting this variation among species. Furthermore, species with higher long-term effective population sizes tend to have lower mutation rates per generation, providing support for the drift barrier hypothesis3. The exceptionally high yearly mutation rates of domesticated animals, which have been continually selected on fecundity traits including shorter generation times, further support the importance of generation time in the evolution of mutation rates. Overall, our comparative analysis of pedigree-based mutation rates provides ecological insights on the mutation rate evolution in vertebrates.

Sea cucumbers (Holothuroidea) are a diverse clade of echinoderms found from intertidal waters to the bottom of the deepest trenches. Their reduced skeletons and limited number of phylogenetically-informative traits have long obfuscated morphological classifications. Sanger-sequenced molecular datasets have also failed to constrain the position of major lineages. Noteworthy, topological uncertainty has hindered a resolution for Neoholothuriida, a highly diverse clade of Permo-Triassic age. We perform the first phylogenomic analysis of Holothuroidea, combining existing datasets with twelve novel transcriptomes. Using a highly-curated dataset of 1,100 orthologues, our efforts recapitulate previous results, struggling to resolve interrelationships among neoholothuriid clades. Three approaches to phylogenetic reconstruction (concatenation under both site-homogeneous and site-heterogeneous models, and coalescent-aware inference) result in alternative resolutions, all of which are recovered with strong support, and across a range of datasets filtered for phylogenetic usefulness. We explore this intriguing result using gene-wise log-likelihood scores, and attempt to correlate these with a large set of gene properties. While presenting novel ways of exploring and visualizing support for alternative trees, we are unable to discover significant predictors of topological preference, and our efforts fail to favor one topology. Neoholothuriid genomes seem to retain an amalgam of signals derived from multiple phylogenetic histories.

The recurrent evolution of resistance to cardiotonic steroids (CTS) across diverse animals most frequently involves convergent amino-acid substitutions to the H1-H2 extracellular loop of Na+,K+-ATPase (NKA). Previous work established that hystricognath rodents (e.g. chinchilla) and pterocliform birds (sandgrouse) have convergently evolved amino-acid insertions in the H1-H2 loop, but their functional significance is not known. Using protein engineering, we show that these insertions have distinct effects on CTS resistance of NKA in the two lineages that strongly depend on intramolecular interactions with other residues. Removing the insertion in the chinchilla lineage unexpectedly increases CTS resistance and decreases NKA activity. In the sandgrouse lineage, the insertion works in concert with the substitution Q111R to increase CTS resistance while maintaining wild-type ATPase activity levels. Molecular docking simulations provide additional insight into the biophysical mechanisms responsible for the context-specific CTS insensitivity of the enzyme. Our results highlight the diversity of genetic substrates that underlie CTS insensitivity in vertebrate NKA and reveal how amino-acid insertions can alter the phenotypic effects of point mutations at key sites in the same protein domain.

The controversy over the taxonomic identity of the eggs exploited by Australia’s first people around 50,000 y ago is resolved. The birds that laid these eggs are extinct, and distinguishing between two main candidates, a giant flightless “mihirung” Genyornis and a large megapode Progura, had proven impossible using morphological and geochemical methods. Ancient DNA sequencing remains inconclusive because of the age and burial temperature of the eggshell. In contrast, ancient protein sequences recovered from the eggshell enabled estimation of the evolutionary affinity between the egg and a range of extant taxa. The eggs are those of a Galloanseres (a group that includes extinct Dromornithidae, as well as extant landfowl and waterfowl), Genyornis, and not of the megapode (Megapodiidae, crown Galliformes). The realization that ancient biomolecules are preserved in “fossil” samples has revolutionized archaeological science. Protein sequences survive longer than DNA, but their phylogenetic resolution is inferior; therefore, careful assessment of the research questions is required. Here, we show the potential of ancient proteins preserved in Pleistocene eggshell in addressing a longstanding controversy in human and animal evolution: the identity of the extinct bird that laid large eggs which were exploited by Australia’s indigenous people. The eggs had been originally attributed to the iconic extinct flightless bird Genyornis newtoni (†Dromornithidae, Galloanseres) and were subsequently dated to before 50 ± 5 ka by Miller etnbsp;al. [Nat. Commun. 7, 10496 (2016)]. This was taken to represent the likely extinction date for this endemic megafaunal species and thus implied a role of humans in its demise. A contrasting hypothesis, according to which the eggs were laid by a large mound-builder megapode (Megapodiidae, Galliformes), would therefore acquit humans of their responsibility in the extinction of Genyornis. Ancient protein sequences were reconstructed and used to assess the evolutionary proximity of the undetermined eggshell to extant birds, rejecting the megapode hypothesis. Authentic ancient DNA could not be confirmed from these highly degraded samples, but morphometric data also support the attribution of the eggshell to Genyornis. When used in triangulation to address well-defined hypotheses, paleoproteomics is a powerful tool for reconstructing the evolutionary history in ancient samples. In addition to the clarification of phylogenetic placement, these data provide a more nuanced understanding of the modes of interactions between humans and their environment.

SignificanceThe controversy over the taxonomic identity of the eggs exploited by Australia's first people around 50,000 y ago is resolved. The birds that laid these eggs are extinct, and distinguishing between two main candidates, a giant flightless "mihirung"

The recurrent evolution of resistance to cardiotonic steroids (CTS) across diverse animals most frequently involves convergent amino acid substitutions in the H1-H2 extracellular loop of Na+,K+-ATPase (NKA). Previous work revealed that hystricognath rodents (e.g., chinchilla) and pterocliform birds (sandgrouse) have convergently evolved amino acid insertions in the H1-H2 loop, but their functional significance was not known. Using protein engineering, we show that these insertions have distinct effects on CTS resistance in homologs of each of the two species that strongly depend on intramolecular interactions with other residues. Removing the insertion in the chinchilla NKA unexpectedly increases CTS resistance and decreases NKA activity. In the sandgrouse NKA, the amino acid insertion and substitution Q111R both contribute to an augmented CTS resistance without compromising ATPase activity levels. Molecular docking simulations provide additional insight into the biophysical mechanisms responsible for the context-specific mutational effects on CTS insensitivity of the enzyme. Our results highlight the diversity of genetic substrates that underlie CTS insensitivity in vertebrate NKA and reveal how amino acid insertions can alter the phenotypic effects of point mutations at key sites in the same protein domain.

Aciculata (Eunicida + Phyllodocida) is among the largest clades of annelids, comprising almost half of the known diversity of all marine annelids. Despite the group's large size and biological importance, most phylogenomic studies on Annelida to date have had a limited sampling of this clade. The phylogenetic placement of many clades within Phyllodocida in particular has remained poorly understood. To resolve the relationships within Aciculata we conducted a large-scale phylogenomic analysis based on 24 transcriptomes (13 new), chosen to represent many family-ranked taxa that have never been included in a broad phylogenomic study. Our sampling also includes several enigmatic taxa with challenging phylogenetic placement, such as Histriobdella, Struwela, Lacydonia, Pilargis and the holopelagic worms Lopadorrhynchus, Travisiopsis and Tomopteris. Our robust phylogeny allows us to name and place some of these problematic clades and has significant implications on the systematics of the group. Within Eunicida we reinstate the names Eunicoidea and Oenonoidea. Within Phyllodocida we delineate Phyllodociformia, Glyceriformia, Nereidiformia, Nephtyiformia and Aphroditiformia. Phyllodociformia now includes: Lacydonia, Typhloscolecidae, Lopadorrhynchidae and Phyllodocidae. Nephtyiformia includes Nephtyidae and Pilargidae. We also broaden the delineation of Glyceriformia to include Sphaerodoridae, Tomopteridae and Glyceroidea (Glyceridae + Goniadidae). Furthermore, our study demonstrates and explores how conflicting, yet highly supported topologies can result from confounding signals in gene trees.

The Xerces Blue (Glaucopsyche xerces) is considered to be the first butterfly to become extinct at global scale in historical times. It was notable for its chalky lavender wings with conspicuous white spots on the ventral wings. The last individuals were collected in their restricted habitat, in the dunes near the Presidio military base in San Francisco, in 1941. To explore the demographic history of this iconic butterfly and to better understand why it went extinct, we sequenced at medium coverage the genomes of four 80 to 100-year-old Xerces Blue specimens and seven historic specimens of its closest relative, the Silvery Blue (G. lygdamus). We compared these to a novel annotated genome of the Green-Underside Blue (G. alexis). Phylogenetic relationships inferred from complete mitochondrial genomes indicate that Xerces Blue was a distinct species that diverged from the Silvery Blue lineage at least 850,000 years ago. Using nuclear genomes, we show that both species experienced population growth during the MIS 7 interglacial period, but the Xerces Blue decreased to a very low effective population size subsequently, a trend opposite to that observed in the Silvery Blue. Runs of homozygosity in the Xerces Blue were significantly greater than in the Silvery Blue, suggesting a higher incidence of inbreeding. In addition, the Xerces Blue carried a higher proportion of derived, putatively deleterious amino acid-changing alleles than the Silvery Blue. These results demonstrate that the Xerces Blue experienced more than 100 thousand years of population decline, prior to its human-induced final extinction.

Background During evolutionary history, molecular mechanisms have emerged to cope with deleterious mutations. Frameshift insertions in protein-coding sequences are extremely rare because they disrupt the reading frame. There are a few known examples of their correction through translational frameshifting, a process that enables ribosomes to skip nucleotides during translation to regain proper reading frame. Corrective frameshifting has been proposed to act on the single base pair insertion at position 174 of the mitochondrial NADH dehydrogenase subunit 3 gene (ND3) that has been observed in several turtles and birds. However, the relatively sparse taxonomic representation has hampered our understanding of the evolution of this insertion in vertebrates. Results Here, we analyzed 87,707 ND3 sequences from 10,309 vertebrate taxa to reveal the evolutionary history of this insertion and its common genomic characteristics. We confirmed that the insertion only appears in turtles and birds and reconstructed that it evolved independently in both groups with complex patterns of gains and losses. The insertion was observed in almost all bird orders but was absent in all members of the diverse Passeriformes. We found strong conservation in the nucleotides surrounding the insertion in both turtles and birds, which implies that the insertion enforces structural constraints that could be involved in its correction. Conclusions Our study demonstrates that frameshifts can be widespread and can be retained for millions of years if they are embedded in a conserved sequence theme.

Whole-genome sequencing projects are increasingly populating the tree of life and characterizing biodiversity1–4. Sparse taxon sampling has previously been proposed to confound phylogenetic inference5, and captures only a fraction of the genomic diversity. Here we report a substantial step towards the dense representation of avian phylogenetic and molecular diversity, by analysing 363 genomes from 92.4% of bird families—including 267 newly sequenced genomes produced for phase II of the Bird 10,000 Genomes (B10K) Project. We use this comparative genome dataset in combination with a pipeline that leverages a reference-free whole-genome alignment to identify orthologous regions in greater numbers than has previously been possible and to recognize genomic novelties in particular bird lineages. The densely sampled alignment provides a single-base-pair map of selection, has more than doubled the fraction of bases that are confidently predicted to be under conservation and reveals extensive patterns of weak selection in predominantly non-coding DNA. Our results demonstrate that increasing the diversity of genomes used in comparative studies can reveal more shared and lineage-specific variation, and improve the investigation of genomic characteristics. We anticipate that this genomic resource will offer new perspectives on evolutionary processes in cross-species comparative analyses and assist in efforts to conserve species., A dataset of the genomes of 363 species from the Bird 10,000 Genomes Project shows increased power to detect shared and lineage-specific variation, demonstrating the importance of phylogenetically diverse taxon sampling in whole-genome sequencing.

New genome assemblies have been arriving at a rapidly increasing pace, thanks to decreases in sequencing costs and improvements in third-generation sequencing technologies1–3. For example, the number of vertebrate genome assemblies currently in the NCBI (National Center for Biotechnology Information) database4 increased by more than 50% to 1,485 assemblies in the year from July 2018 to July 2019. In addition to this influx of assemblies from different species, new human de novo assemblies5 are being produced, which enable the analysis of not only small polymorphisms, but also complex, large-scale structural differences between human individuals and haplotypes. This coming era and its unprecedented amount of data offer the opportunity to uncover many insights into genome evolution but also present challenges in how to adapt current analysis methods to meet the increased scale. Cactus6, a reference-free multiple genome alignment program, has been shown to be highly accurate, but the existing implementation scales poorly with increasing numbers of genomes, and struggles in regions of highly duplicated sequences. Here we describe progressive extensions to Cactus to create Progressive Cactus, which enables the reference-free alignment of tens to thousands of large vertebrate genomes while maintaining high alignment quality. We describe results from an alignment of more than 600 amniote genomes, which is to our knowledge the largest multiple vertebrate genome alignment created so far., The Progressive Cactus program can create reference-free alignments of hundreds of large vertebrate genomes efficiently, and is used for the alignment of more than 600 amniote genomes.

During the Last Glacial Maximum (LGM), global sea levels were 120-130 m lower than today, resulting in the emergence of most continental shelves and extirpation of subtidal organisms from these areas. During the interglacial periods, rapid inundation of shelf regions created a dynamic environment for coastal organisms, such as the charismatic leafy seadragon (Phycodurus eques, Syngnathidae), a brooder with low dispersal ability inhabiting kelp beds in temperate Australia. Reconstructions of the palaeoshoreline revealed that the increase of shallow areas since the LGM was not uniform across the species' range and we investigated the effects of these asymmetries on genetic diversity and structuring. Using targeted capture of 857 variable ultraconserved elements (UCEs, 2,845 single nucleotide polymorphisms) in 68 individuals, we found that the regionally different shelf topographies were paralleled by contrasting population genetic patterns. In the west, populations may not have persisted through sea-level lows because shallow seabed was very limited. Shallow genetic structure, weak expansion signals and a westward cline in genetic diversity indicate a postglacial recolonization of the western part of the range from a more eastern location following sea-level rise. In the east, shallow seabed persisted during the LGM and increased considerably after the flooding of large bays, which resulted in strong demographic expansions, deeper genetic structure and higher genetic diversity. This study suggests that postglacial flooding with rising sea levels produced locally variable signatures in colonizing populations.

Sabellida is a well-known clade containing tube-dwelling annelid worms with a radiolar crown. Iterative phylogenetic analyses over three decades have resulted in three main clades being recognized; Fabriciidae, Serpulidae and Sabellidae, with Fabriciidae proposed as the sister group to Serpulidae. However, relationships within Sabellidae have remained poorly understood, with a proliferation of genera. In order to obtain a robust phylogeny with optimal support, we conducted a large-scale phylogenomic analysis with 19 new sabellid transcriptomes for a total of 21 species. In contrast to earlier findings based on limited DNA data, our results support the position of Fabriciidae as sister taxon to a Sabellidae + Serpulidae clade. Our large sampling within Sabellidae also allows us to establish a stable phylogeny within this clade. We restrict Sabellinae to a subclade of Sabellidae and broaden the previously monotypic Myxicolinae to include Amphicorina and Chone. We tested the robustness of species tree reconstruction by subsampling increasing numbers of genes to uncover hidden support of alternative topologies. Our results show that inclusion of more genes leads to a more stable topology with higher support, and also that including higher divergence genes leads to stronger resolution.

Terebelliformia—“spaghetti worms” and their allies—are speciose and ubiquitous marine annelids but our understanding of how their morphological and ecological diversity evolved is hampered by an uncertain delineation of lineages and their phylogenetic relationships. Here, we analyzed transcriptomes of 20 terebelliforms and an outgroup to build a robust phylogeny of the main lineages grounded on 12,674 orthologous genes. We then supplemented this backbone phylogeny with a denser sampling of 121 species using five genes and 90 morphological characters to elucidate fine-scale relationships. The monophyly of six major taxa was supported: Pectinariidae, Ampharetinae, Alvinellidae, Trichobranchidae, Terebellidae and Melinninae. The latter, traditionally a subfamily of Ampharetidae, was unexpectedly the sister to Terebellidae, and hence becomes Melinnidae, and Ampharetinae becomes Ampharetidae. We found no support for the recently proposed separation of Telothelepodidae, Polycirridae and Thelepodidae from Terebellidae. Telothelepodidae was nested within Thelepodinae and is accordingly made its junior synonym. Terebellidae contained the subfamily-ranked taxa Terebellinae and Thelepodinae. The placement of the simplified Polycirridae within Terebellinae differed from previous hypotheses, warranting the division of Terebellinae into Lanicini, Procleini, Terebellini and Polycirrini. Ampharetidae (excluding Melinnidae) were well-supported as the sister group to Alvinellidae and we recognize three clades: Ampharetinae, Amaginae and Amphicteinae. Our analysis found several paraphyletic genera and undescribed species. Morphological transformations on the phylogeny supported the hypothesis of an ancestor that possessed both branchiae and chaetae, which is at odds with proposals of a “naked” ancestor. Our study demonstrates how a robust backbone phylogeny can be combined with dense taxon coverage and morphological traits to give insights into the evolutionary history and transformation of traits.

In Dixie Valley, Nevada, an isolated population of toads has been the subject of proactive conservation measures by the Nevada Department of Wildlife and the U.S. Fish and Wildlife Service since 2008 due to concerns about potential habitat degradation resulting from exploitation of nearby geothermal energy resources. These toads appear to belong within the Anaxyrus boreas species group but are commonly referred to as Dixie Valley toads (DVTs). The DVT is currently confined to an extremely narrow habitat range (370 ha) that is geographically isolated from any other A. boreas population. In this study, genetic variations in mitochondrial genes and 11 microsatellite loci were used to assess the affinities of DVTs in relation to members of the A. boreas species group. We compared results from DVTs with previously published data spanning much of the range of A. boreas in the United States and new data from a nearby toad population within Dixie Valley. Data from both mitochondrial DNA and microsatellites placed DVTs inside the A. boreas species group. In particular, DVTs fell into a cluster of A. boreas from Washington and California, along with other species from the A. boreas species group, namely A. nelsoni, A. canorus, and A. exsul. Genetic differentiation of DVTs was lowest between A. boreas populations in Washington and California. However, allele frequencies were significantly different between DVTs and all other populations, including a nearby locality within Dixie Valley. This genetic differentiation, along with the DVT's geographical isolation and restricted habitat, warrants recognition of the DVT as a distinct management unit.

Cactus, a reference-free multiple genome alignment program, has been shown to be highly accurate, but the existing implementation scales poorly with increasing numbers of genomes, and struggles in regions of highly duplicated sequence. We describe progressive extensions to Cactus that enable reference-free alignment of tens to thousands of large vertebrate genomes while maintaining high alignment quality. We show that Cactus is capable of scaling to hundreds of genomes and beyond by describing results from an alignment of over 600 amniote genomes, which is to our knowledge the largest multiple vertebrate genome alignment yet created. Further, we show improvements in orthology resolution leading to downstream improvements in annotation.

Birds are a group with immense availability of genomic resources, and hundreds of forthcoming genomes at the doorstep. We review recent developments in whole genome sequencing, phylogenomics, and comparative genomics of birds. Short read based genome assemblies are common, largely due to efforts of the Bird 10K genome project (B10K). Chromosome-level assemblies are expected to increase due to improved long-read sequencing. The available genomic data has enabled the reconstruction of the bird tree of life with increasing confidence and resolution, but challenges remain in the early splits of Neoaves due to their explosive diversification after the Cretaceous-Paleogene (K-Pg) event. Continued genomic sampling of the bird tree of life will not just better reflect their evolutionary history but also shine new light onto the organization of phylogenetic signal and conflict across the genome. The comparatively simple architecture of avian genomes makes them a powerful system to study the molecular foundation of bird specific traits. Birds are on the verge of becoming an extremely resourceful system to study biodiversity from the nucleotide up.

As the only endemic neotropical parrot to have recently lived in the northern hemisphere, the Carolina parakeet (Conuropsis carolinensis) was an iconic North American bird. The last surviving specimen died in the Cincinnati Zoo in 1918 [1]. The cause of its extinction remains contentious: besides excessive mortality associated to habitat destruction and active hunting, their survival could have been negatively affected by its range having become increasingly patchy [2] or by the exposure to poultry pathogens [3, 4]. In addition, the Carolina parakeet showed a predilection for cockleburs, an herbaceous plant that contains a powerful toxin, carboxyatractyloside, or CAT [5], which did not seem to affect them but made the birds notoriously toxic to most predators [3]. To explore the demographic history of this bird, we generated the complete genomic sequence of a preserved specimen held in a private collection in Espinelves (Girona, Spain), as well as of a close extant relative, Aratinga solstitialis. We identified two non-synonymous genetic changes in two highly conserved proteins known to interact with CAT that could underlie a specific dietary adaptation to this toxin. Our genomic analyses did not reveal evidence of a dramatic past demographic decline in the Carolina parakeet; also, its genome did not exhibit the long runs of homozygosity that are signals of recent inbreeding and are typically found in endangered species. As such, our results suggest its extinction was an abrupt process and thus likely solely attributable to human causes. Gelabert et al. report the whole genome of the extinct Carolina parakeet and provide evidence of its phylogeny, adaptation to a toxic cocklebur diet, and demographic history. The lack of signs of recent inbreeding typically found in endangered species suggests its abrupt extinction was human mediated., This work was supported by ERC Consolidator Grant 681396 “Extinction Genomics” to M.T.P.G. and M.S.-V., Obra Social ‘‘La Caixa’’ and Secretaria d’Universitats i Recerca ( GRC2017-SGR880 ) to T.M.-B. and C.L.-F., BFU2017-86471-P and PGC2018-101927-B-I00 from MINECO/FEDER, UE to T.M.-B, and PGC2018-095931-B-100 from MINECO/FEDER, UE to C.L.-F. T.M.-B. is also supported by a U01 MH106874 grant and Howard Hughes International Early Career and CERCA Programme (Generalitat de Catalunya). A.A. is partially supported by the Strategic Funding UID/Multi/04423/2019 through national funds provided by the Portuguese Foundation for Science and Technology (FCT) and the European Regional Development Fund —program PT2020 , by the European Structural and Investment Funds —Competitiveness and Internationalization Operational Program-COMPETE 2020, and by National Funds through the FCT under the project PTDC/CTA-AMB/31774/2017(POCI-01-0145-FEDER/031774/2017 ).

In Supplementary Table 1 of this Article, 23 samples (B10K-DU-029-32, B10K-DU-029-33, B10K-DU-029-36 to B10K-DU-029-44, B10K-DU- 029-46, B10K-DU-029-47, B10K-DU-029-49 to B10K-DU-029-53, B10K-DU- 029-75 to B10K-DU-029-77, B10K-DU-029-80, and B10K-DU-030-03; styled in boldface in the revised table) were assigned to the incorrect institution. Supplementary Table 1 has been amended to reflect the correct source institution for these samples, and associated data (tissue, museum ID/source specimen ID, site, state/province, latitude, longitude, date collected and sex) have been updated accordingly. The original table is provided as Supplementary Information to this Amendment, and the original Article has been corrected online.

The common seadragon is an iconic fish with presumed limited dispersal, because juveniles hatch directly from the tail of the male parent. Nothing is presently known of their phylogeographic structure, despite conservation concerns and a distribution spanning southern Australia. Here, we sequenced mitochondrial genes from 201 common seadragons in Western Australia, South Australia, Victoria, Tasmania and New South Wales. We show that common seadragon populations are highly structured geographically, and that genetic variation varies significantly. The historical Bassian Isthmus appears to have left a strong imprint on population structure. Populations east of the Bassian Isthmus are low in diversity and appear more connected than those in the west, although this is likely caused by a recent expansion from a common source after the Last Glacial Maximum. All individuals from Eden, Bicheno and Hobart are represented by only two haplotypes. Populations west of the Bassian Isthmus are more diverse, with the highest diversity indices shown by Western Australian and Spencer Gulf populations. A large sampling gap across the Great Australian Bight is yet to be resolved; the west versus east break here may be an artifact of this gap. Almost all sampled populations can be inferred to have limited gene flow among them, which has implications for recovery after local extinction. Populations thought to be in decline (Sydney, Hobart) showed low genetic diversity, which may make them vulnerable to further reductions.

Summary The Indo-Pacific humpback dolphin (Sousa chinensis) is a small inshore species of odontocete cetacean listed as Vulnerable on the IUCN Red List. Here, we report on the evolution of S. chinensis chromosomes from its cetruminant ancestor and elucidate the evolutionary history and population genetics of two neighboring S. chinensis populations. We found that breakpoints in ancestral chromosomes leading to S. chinensis could have affected the function of genes related to kidney filtration, body development, and immunity. Resequencing of individuals from two neighboring populations in the northwestern South China Sea, Leizhou Bay and Sanniang Bay, revealed genetic differentiation, low diversity, and small contemporary effective population sizes. Demographic analyses showed a marked decrease in the population size of the two investigated populations over the last ~4,000 years, possibly related to climatic oscillations. This study implies a high risk of extinction and strong conservation requirement for the Indo-Pacific humpback dolphin., Graphical Abstract, Highlights • Deducing chromosome evolution from ancestral Cetruminantia and ancestral Odontoceti • Reconstructing the demographic history of Sousa chinensis • Implying high risk of extinction and strong conservation requirement for S. chinensis, Evolutionary Biology; Genetics; Genomics

Background A range of higher animal taxa are shared across various chemosynthesis-based ecosystems (CBEs), which demonstrates the evolutionary link between these habitats, but on a global scale the number of species inhabiting multiple CBEs is low. The factors shaping the distributions and habitat specificity of animals within CBEs are poorly understood, but geographic proximity of habitats, depth and substratum have been suggested as important. Biogeographic studies have indicated that intermediate habitats such as sedimented vents play an important part in the diversification of taxa within CBEs, but this has not been assessed in a phylogenetic framework. Ampharetid annelids are one of the most commonly encountered animal groups in CBEs, making them a good model taxon to study the evolution of habitat use in heterotrophic animals. Here we present a review of the habitat use of ampharetid species in CBEs, and a multi-gene phylogeny of Ampharetidae, with increased taxon sampling compared to previous studies. Results The review of microhabitats showed that many ampharetid species have a wide niche in terms of temperature and substratum. Depth may be limiting some species to a certain habitat, and trophic ecology and/or competition are identified as other potentially relevant factors. The phylogeny revealed that ampharetids have adapted into CBEs at least four times independently, with subsequent diversification, and shifts between ecosystems have happened in each of these clades. Evolutionary transitions are found to occur both from seep to vent and vent to seep, and the results indicate a role of sedimented vents in the transition between bare-rock vents and seeps. Conclusion The high number of ampharetid species recently described from CBEs, and the putative new species included in the present phylogeny, indicates that there is considerable diversity still to be discovered. This study provides a molecular framework for future studies to build upon and identifies some ecological and evolutionary hypotheses to be tested as new data is produced. Electronic supplementary material The online version of this article (10.1186/s12862-017-1065-1) contains supplementary material, which is available to authorized users.

Until recently, only two species of seadragon were known, Phycodurus eques (the leafy seadragon) and Phyllopteryx taeniolatus (the common seadragon), both from Australia. In 2015, we described a new species of seadragon, Phyllopteryx dewysea (the ruby seadragon). Although the leafy and common seadragons are well known and commonly seen in aquarium exhibits world-wide, the ruby seadragon was known only from four preserved specimens, leaving many aspects of its biology unknown. Based on specimen records, it was speculated that the ruby seadragon normally lives at depths beyond recreational SCUBA diving limits, which may also explain why it went undiscovered for so long. The ruby seadragon also bears a superficial resemblance to the common seadragon, with a number of specimens misidentified in museum collections. The only recent live-collected specimen was trawled from the Recherche Archipelago, a cluster of over 100 islands in Western Australia. We took a small remotely operated vehicle (miniROV) to this locality to obtain the first images of live ruby seadragons. We made observations on the seadragon habitat and behavior, including feeding. We also provide new key observations on their morphology, notably that they lack dermal appendages and have a prehensile tail. We recommend that the ruby seadragon be protected as soon as practicable.

The exploration of Earth's biodiversity is an exciting and ongoing endeavour. Here, we report a new species of seadragon from Western Australia with substantial morphological and genetic differences to the only two other known species. We describe it as Phyllopteryx dewysea n. sp. Although the leafy seadragon ( Phycodurus eques ) and the common seadragon ( Phyllopteryx taeniolatus ) occur along Australia's southern coast, generally among relatively shallow macroalgal reefs, the new species was found more offshore in slightly deeper waters. The holotype was trawled east of the remote Recherche Archipelago in 51 m; additional specimens extend the distribution west to Perth in 72 m. Molecular sequence data show clear divergence from the other seadragons (7.4–13.1% uncorrected divergence in mitochondrial DNA) and support a placement as the sister-species to the common seadragon. Radiographs and micro-computed tomography were used on the holotype of the new species and revealed unique features, in addition to its unusual red coloration. The discovery provides a spectacular example of the surprises still hidden in our oceans, even in relatively shallow waters.

Amphisamytha has five currently recognized species. One of these, A. galapagensis has been reported from numerous hydrothermal vents and methane seeps across the Pacific Ocean. Here, a collection of Amphisamytha from a range of Pacific habitats, as well as Amathys lutzi from Atlantic hydrothermal vents, were studied using morphology and DNA sequences from mitochondrial (COI and 16S rRNA) and nuclear (18S rRNA) genes. Phylogenetic analyses revealed a deep-sea clade comprised of the previously recognized Amphisamytha species, and three lineages that are treated as new species. The morphologically distinct Amathys lutzi was nested within Amphisamytha and the monotypic Amathys is treated as a junior synonym of Amphisamytha, with a resulting name change to Amphisamytha lutzi comb. nov. A revision of the other Amphisamytha species, the description of three new species, and a key are provided. The geographic range of the cosmopolitan' A. galapagensis is restricted to hydrothermal vents of the East Pacific Rise and the Galapagos Ridge. Amphisamytha fauchaldi, previously only known from sedimented hydrothermal vents of the Guaymas Basin (Gulf of California, Mexico), is recorded from cold seeps off the coasts of Costa Rica and Oregon. Amphisamytha carldarei sp. nov. is described from hydrothermal vents of the northeast Pacific (Juan de Fuca), while A. jacksoni sp. nov. spans much of the East Pacific Rise and is sympatric with A. galapagensis for part of its range. Amphisamytha julianeae sp. nov. is found at western Pacific vent systems in sympatry with A. vanuatuensis. Relaxed molecular clock analyses were calibrated using the vicariant event involving the Farallon-Pacific Ridge in the northern Pacific Ocean. This provides mean dates for the origin of the deep-sea Amphisamytha clade at 44 or 55 million years ago, and the separation of the PacificAtlantic sister-species pair at 13 or 21 million years ago, depending on the tree used.

The leafy seadragon (Phycodurus eques, Syngnathidae), is a charismatic endemic of Australia’s temperate coast. The species exhibits remarkable camouflage in its kelp and seagrass habitat. These habitats have been retreating throughout the range of the species, leading to concerns about the persistence of leafy seadragons. Despite being a popular aquarium display and a flagship for coastal conservation, little is known about leafy seadragon biology. We used 7 microsatellite markers and 2 mitochondrial DNA fragments to investigate the population structuring and genetic diversity of 71 individuals. Bayesian cluster analysis identified 2 main genetic partitions, one in Western Australia and the other in South Australia. Shallower, yet significant, differentiation of mitochondrial DNA (φST) and microsatellites (FST, F′ST, D) was found on the smaller geographic scales in South Australia. Distinct groups were supported in Encounter Bay, on both shores of Gulf St Vincent, and in Spencer Gulf. Sample sizes were too small in the eastern Great Australian Bight and in the westernmost locality to address genetic differentiation in these regions. Overall genetic diversity was moderate to low, but particularly low levels were found in the Western Australian cluster. This latter point needs confirmation at other sites in Western Australia. In South Australia, signatures of demographic changes were detected, which may have been caused by a population expansion due to post-glacial reflooding of the gulfs. The Western Australian and South Australian clusters appeared as demographically independent units. Conservation actions should focus on preserving genetic diversity at local scales and maintaining habitat connectivity.

Plant anti-herbivore defence is inducible by both insect feeding and egg deposition. However, little is known about the ability of insect eggs to induce defences directed not against the eggs themselves, but against larvae that subsequently hatch from the eggs. We studied how oviposition (OP) by the sawflyDiprion pinionPinus sylvestrisfoliage affects the plant's defensive potential against sawfly larvae. Larvae that initiated their development onP. sylvestristwigs on which they hatched from eggs gained less weight and suffered higher mortality than those fed on egg-free twigs. The poor performance of these larvae also affected the next herbivore generation since fecundity of resulting females was lower than that of females which spent their larval development on egg-free pine. Transcript levels ofP. sylvestrissesquiterpene synthases (PsTPS1,PsTPS2) were increased byD. piniOP, reached their highest levels just before larval hatching, and decreased when larvae started to feed. However, concentrations of terpenoid and phenolic metabolites presumed to act as feeding deterrents or toxins for herbivores did not change significantly after OP and feeding. Nevertheless, our performance data suggest that insect egg deposition may act to ‘warn’ a plant of upcoming feeding damage by larvae.